INVASION OF EPITHELIAL-CELLS BY SHIGELLA-FLEXNERI INDUCES TYROSINE PHOSPHORYLATION OF CORTACTIN BY A PP60(C-SRC)-MEDIATED SIGNALING PATHWAY

Shigella flexneri causes bacillary dysentery in humans by invading epi thelial cells of the colon. Cell invasion occurs via bacterium-directe d phagocytosis, a process requiring polymerization of actin at the sit e of bacterial entry, We show that invasion of HeLa cells by S.flexner i induces tyrosine phosphorylation of cortactin, a host cell protein p reviously identified as a cytoskeleton-associated protein tyrosine kin ase (PTK) substrate for the proto-oncoprotein pp60(c-src). Immunolocal ization experiments indicate that cortactin is recruited to submembran ous actin filaments formed during bacterial entry, In particular, cort actin is highly enriched in membrane ruffles of the entry structure, w hich engulf entering bacteria, and also in the periphery of the phagos ome early after bacterial internalization. The proto-oncoprotein pp60( c-src) appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the l evel of cortactin tyrosine phosphorylation induced during bacterial en try, Immunolocalization studies in pp60(c-src)-overexpressing HeLa cel ls indicate that pp60(c-src) is, recruited to the entry structure and to the periphery of the phagosome, where pp60(c-src) appears to accumu late in association with the membrane, Our results suggest that epithe lial cell invasion by S.flexneri involves recruitment and kinase activ ation of pp60(c-src). Signalling by the protooncoprotein pp60(c-src) m ay play a role in cytoskeletal changes that facilitate S.flexneri upta ke into epithelial cells, since transient overexpression of pp60(c-src ) in HeLa cells can provoke membrane ruffling and appears also to stim ulate bacterial uptake of a non-invasive S.flexneri strain.